Diagnostic evaluation of antibody responses to commonly recognized prostate cancer-associated antigens

ABSTRACT

The present invention relates to a plurality of antigens that together form a panel of immunoreactive molecules suitable for identifying candidates for prostate cancer examination. Methods for identifying antibodies indicative of a pre-malignant or malignant prostate are disclosed. Further disclosed are kits that can be used to practice the above methods.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.14/757,607, filed Dec. 23, 2015, now U.S. Pat. No. 10,191,056 which is acontinuation of U.S. application Ser. No. 13/088,594, filed Apr. 18,2011, now abandoned which claims the benefit of U.S. Provisional PatentApplication No. 61/324,953, filed Apr. 16, 2010, each of which isincorporated herein by reference as if set forth in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under RR016489 awardedby the National Institutes of Health. The government has certain rightsin the invention.

SEQUENCE LISTING

A sequence listing is submitted in PDF and text formats on one CompactDisc-Read Only Memory (CD-ROM), in duplicate, and is incorporated hereinby reference. “Copy 1” of the CD-ROM contains Seq List960296_01933_ST25.PDF, created Dec. 23, 2015, 1620 KB and Seq List960296_01933_ST25.TXT, created Dec. 23, 2015, 2695 KB. “Copy 2” of theCD-ROM contains Seq List 960296_01933_ST25.PDF, created Dec. 23, 2015,1620 KB and Seq List 960296_01933_ST25.TXT, created Dec. 23, 2015, 2695KB.

BACKGROUND

Prostate cancer is prevalent worldwide. Men living in the United Stateshave approximately a 1 in 6 chance of being diagnosed with prostatecancer at some point during their lifetime, and approximately a 1 in 50chance of dying from prostate cancer (Jemal et al., CA Cancer J. Clin.59:225-249 (2009), incorporated herein by reference as if set forth inits entirety). The number of men afflicted with prostate cancer isincreasing rapidly as the population of males over the age of 50 grows.Thus, strategies for detecting prostate cancer in its early stages areurgently needed.

Conventional prostate cancer screening methods, including digital rectalexamination, transrectal ultrasound, and prostate specific antigen (PSA)testing, lack sensitivity and specificity. Elements of PSA screening arecontroversial: practitioners disagree on the best age for screening, howto interpret elevated PSA values, and how to identify prostate cancersin patients with low serum PSA values. There are also concerns that PSAscreening may lead to unnecessary interventions that do not affectlong-term outcome and survival (Greene et al., J Urol 182:2232-2241(2009), incorporated herein by reference as if set forth in itsentirety). To overcome the disadvantages of prostate cancer screeningmethods, researchers have attempted to identify antigens produced bytumor cells that can be utilized as prostate cancer markers. Serum-basedscreening methods have allowed for rapid identification of many tumorantigens in individuals with many different tumor types (Sahin et al.,Proc. Nat. Acad. Sci. 92:11810-11813(1995), incorporated herein byreference as if set forth in its entirety). In fact, serum screening hasbeen so successful that literally hundreds of cancer-associatedantigenic proteins have been identified.

Serological identification of antigens by recombinant expression cloning(SEREX) has been used to prioritize potential vaccine targets forprostate cancer. Immunologically recognized proteins of the prostatehave been identified by screening sera from: i) individuals withprostate cancer (Dunphy et al., Update Canc. Ther. 22:273-284 (2006);Fossa et al., Br. J. Cancer 83:743-749 (2000); Mooney et al., Int. J.Urol. 13:211-217 (2006)); ii) prostate cancer individuals treated withimmune-active therapies (Dunphy et al., J. Immunother. 28:268-275(2005)); and iii) prostate cancer individuals treated with standardandrogen deprivation therapy (Morse and McNeel, Hum. Immunol. 71:496-504(2010)); each of the foregoing references are herein incorporated byreference as if set forth in their entirety). Sera from individuals withprostate cancer were also used to directly screen a testis tissue cDNAexpression library and a panel of defined cancer-testis antigensaberrantly expressed in solid tumors of different histologic types(Hoeppner et al., Cancer Immun. 6:1-7 (2006); Dubovsky and McNeel,Prostate 67:1781-1790 (2007), each incorporated herein by reference asif set forth in its entirety).

Chronic inflammation has been implicated in lung and colon cancers andmight similarly promote the development of prostate cancers (Palapattuet al., Carcinogenesis 26:1170-1181 (2005); Narayanan et al., Prostate69:133-141 (2009); McDowell et al., Prostate 70:377-389 (2010); Denniset al., Urology 60:78-83 (2002); each incorporated herein by referenceas if set forth in its entirety). Tumor-infiltrating lymphocytes areobserved in prostate tumor specimens, and chronic inflammatory cells(e.g., lymphocytes and mononuclear cells) are observed adjacent to theearliest premalignant lesions of the prostate (De Marzo et al., Am. J.Pathol. 155:1985-1992 (1999) incorporated by reference herein as if setforth in its entirety). In rodents, chronic prostatitis appears topromote the development of prostate tumors (Gilardoni et al., J. Exp.Clin. Cancer Res. 18:493-504 (1999), incorporated by reference herein asif set forth in its entirety). Human prostate-infiltrating lymphocytesobtained at the time of surgery are oligoclonal suggesting that theselymphocytes recognize tissue-specific antigens (Mercader et al., Proc.Nat. Acad. Sci. 98:14565-14570 (2001); Sfanos et al., Prostate69:1694-1703 (2009); each incorporated herein by reference as if setforth in its entirety).

While a number of antigens associated with prostate cancer have beenidentified, it has not yet been determined which of these antigens arepredictive of a malignant or premalignant prostate. This is at least inpart due to the variation in antibody-antigen profiles of individuals;not every antigen associated with prostate cancer or prostatitis isexpressed in every affected individual and not every expressed antigenelicits an immune response in every affected individual. As such, nosingle antigen can serve as a reliable indicator of prostate cancer orprostatitis. The high level of variation of immunoresponsiveness amongindividuals has made it difficult to translate antigens associated withprostate cancer into tools useful for predicting prostate cancer, orpredicting an increased risk of developing prostate cancer, inpopulations of men, wherein populations are defined as groups with orwithout a particular disease or disease stage. Screening men for allantigens that have been associated with prostate cancer iscost-prohibitive and impractical. Thus, there is a need in the art toidentify a subset of antigens specific to antibodies associated withpre-malignant and malignant prostate tissue for identifying candidateindividuals warranting further prostate examination.

BRIEF SUMMARY OF THE INVENTION

The present invention is broadly summarized as relating to a pluralityof antigens that together form a set or panel of immunoreactivemolecules suitable for identifying candidates for prostate cancerexamination. Many cancer detection efforts rely on detecting, withvarying degrees of specificity and sensitivity, cancer-associatedantigens produced by tumor cells. In contrast, the present inventionrelies on detecting immunoresponses to tissue-associated antigens. Thepresent invention contemplates using a plurality of these antigens toidentify antibodies indicative of a pre-malignant or malignant prostate.As used herein, an “antigen” means a substance that when introduced intoan animal stimulates the production of an antibody. An antigen can alsobe a substance produced by the body, wherein an immune response hasbeen, or can be, elicited to that antigen. As used herein, an“antibody,” is a specialized molecule that can specifically bind theantigen that triggered its production.

In a first aspect, the invention is summarized as an antigen panel foridentifying immunoreactivity associated with a premalignant or malignantprostate, the panel comprising a plurality of antigens. As used herein,“immunoreactivity” refers to an animal's immune response to a particularantigen or antigens. One or more of the antigens may be previously knownand identified as being recognized by antibodies in individuals havingprostate cancer and/or prostatitis. In some embodiments of the firstaspect, the panel elicits an immune response in at least about 33% ofmen with prostate cancer or prostatitis. In other embodiments of thefirst aspect, 9% of individuals with prostatitis or prostate cancerexhibit an immune response to at least three of the antigens in thepanel. As use herein, “about” means within 5% of a stated range.

In some embodiments of the first aspect, the plurality of antigens inthe antigen panel is selected from the group consisting of PRO1, PRO2,PRO3, PRO4, PRO5, PRO6, PRO7, PRO8, PRO9, PRO10, PRO11, PRO12, PRO13,PRO14, PRO15, PRO16, PRO17, PRO18, PRO19, PRO20, PRO21, PRO22, PRO23,PRO24, PRO25, PRO26, PRO27, PRO28, PRO29, PRO30, PRO31, PRO32, PRO33,PRO34, PRO35, PRO36, PRO37, PRO38, PRO39, PRO40, PRO41, CTA1, CTA2,CTA3, CTA4, CTA5, CTA6, CTA7, CTA8, CTA9, CTA10, CTA11, CTA12, CTA13,CTA14, CTA15, CTA16, CTA17, CTA18, CTA19, CTA20, CTA21, CTA22, CTA23,CTA24, CTA25, CTA26, CTA27, CTA28, CTA29, PCA1, PCA2, PCA3, PCA4, PCA5,PCA6, PCA7, PCA8, PCA9, PCA10, PCA11, PCA12, PCA13, PCA14, PCA15, PCA16,PCA17, PCA18, PCA19, PCA20, PCA21, PCA22, PCA23, PCA24, PCA25, PCA26,PCA27, PCA28, PCA29, PCA30, ADT1, ADT2, ADT3, ADT4, ADT5, ADT6, ADT7,ADT8, ADT9, ADT10, ADT11, ADT12, ADT13, ADT14, ADT15, ADT16, ADT17,ADT18, ADT19, ADT20, ADT21, ADT22, ADT23, ADT24, ADT25, ADT26, ADT27,and ADT28.

In another embodiment of the first aspect, the plurality of antigens inthe panel includes ADT14, CTA3, CTA23, PCA6, PCA8, PCA10, PCA16, PRO5,PRO10, PRO12, PRO17, PRO18, PRO19, PRO20, PRO24, PRO25, PRO35, CTA27,PCA21, PRO40, PCA2, PRO34, and PRO32.

In a second aspect, the invention relates to a method for identifyinghuman or non-human animals as candidates for prostate cancerexamination. The method includes the steps of contacting an antigenpanel as above with a sample containing at least one antibody specificto at least one antigen of the panel, and observing evidence ofimmunocomplex formation between at least one antibody from the sampleand at least one antigen of the panel. As used herein, “immunocomplex”refers to a complex formed between an antibody and the antigenspecifically recognized by the antibody. An individual is identified asa candidate if at least one immunocomplex forms between an antibody fromthe sample and an antigen in the panel. Samples appropriate for use inthe method include, but are not limited to, tissues, cells, and bodilyfluids, including blood.

In a third aspect, the present invention relates to a kit comprising anantibody detection panel. The kit comprises an antigen panel containinga plurality of antigens as disclosed herein optionally provided in acarrier or medium.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although suitable materials andmethods for the practice or testing of the present invention aredescribed below, other known materials and methods similar or equivalentto those described herein can be used.

Other objectives, advantages and features of the present invention willbecome apparent from the following specification taken in conjunctionwith the accompanying drawings.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1: illustrates high-throughput immunoblot analysis.

FIGS. 2A-2H: illustrate the mean relative density scores for healthymale control blood donors (n=25, open circles) and men withcastrate-resistant prostate cancer (n=25, closed circles). Asterisksdenote significantly higher median reactivity for the cancer populationcompared to the control population.

FIGS. 3A-3F illustrate immunoreactivity of individuals with early andlate stages of prostate cancer. FIG. 3A shows a heatmap analysis of therelative immunoreactivity for individual antigens indicated by spotcolor intensity (immunoreactive: white; intermediate: grey; notimmunoreactive: black). FIGS. 3B-F illustrates a subset analysis for 23antigens with the highest positive likelihood ratio values.

FIGS. 4A-4B illustrate the proportion and standard error of subjects ineach population recognizing at least one (FIG. 4A) or at least three(FIG. 4B) of the antigens identified in Table II. Asterisks denotehigher frequency compared to control population.

FIG. 5: shows a heatmap of the immunoreactivity for individual antigensamong sera samples from male patients with non-prostate tumors. A whitespot indicates a positive score.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention generally relates to a novel panel of a pluralityof antigens for identifying immunoresponses associated with apremalignant or malignant prostate. As used herein, a “set” or a “panel”refers to a group of antigens associated with a premalignant ormalignant prostate. As used herein, “immunoresponse” means theoccurrence of a binding reaction between an antibody specific to anantigen and that antigen. As used herein, a premalignant prostate meansa prostate characterized by tissue that is not yet malignant, but ispoised to become malignant as evidenced by, for example, chronicinflammation or other conditions that can alter cell proliferationand/or cell death. Skilled artisans are familiar with conditions thatare associated with changes in cell proliferation and conditions thatare associated with changes in cell death. As used herein a malignantprostate refers to a prostate containing tumor-forming prostateepithelial cells.

In some embodiments, one or more of the antigens of the panel isassociated with premalignant inflammation of the prostate. As usedherein, “premalignant inflammation” means inflammation, the presence ofwhich indicates an increase in an individual's risk for developing amalignant state or condition. Advantageously, the panel includes aplurality of antigens that make the panel more likely to detectimmunoreactivity when exposed to antibody-containing samples compared tousing a single antigen alone. In some preferred embodiments, antigenshaving higher specificity for prostate cancer are included in the panel,wherein relative specificity is based on receiver operating curveanalysis. In other preferred embodiments, antigens with a positivelikelihood ratio greater than 4 (sensitivity/(1-specificity)) areincluded in the panel. Sensitivity and specificity are based onimmunoreactivity of a population of subjects with prostate diseaserelative to a control population. As used herein, “sensitivity” of anantigen refers to the proportion of positive subjects (i.e., subjectswith prostate cancer or prostatitis) identified as positive by presenceof an immunoresponse to a particular antigen in an assay. As usedherein, “specificity” of an antigen refers to the proportion of negativesubjects (i.e., control subjects without prostate cancer or prostatitis)identified as negative by absence of an immunoresponse to a particularantigen in an assay. The likelihood ratio used to rank antigens is astatistical model used to express how many times more likely a datasetis to fit one statistical model rather than another. For example, anantigen having a likelihood ratio of 4 is 4 times more likely to elicitan immunoresponse in a subject having prostatitis or prostate cancerrelative to a patient lacking prostatitis or prostate cancer. An antigencan be a natural or synthetic protein or fragment thereof,polysaccharide, or nucleic acid. Skilled artisans know that antigens caninduce an immune response and elicit antibody formation. Antibodies canbe molecules synthesized in response to the presence of a foreignsubstance, wherein each antibody has specific affinity for the foreignmaterial that stimulated its synthesis. The specific affinity of anantibody need not be for the entire molecular antigen, but for aparticular site on it called the epitope (Kindt et al., Kuby Immunology,6th Edition 574 pps, (2006), incorporated herein by reference as if setforth in its entirety). Antibodies can be, for example, a natural orsynthetic protein or fragment thereof, or nucleic acids (e.g., aptamers)with protein-binding or other antigen-binding characteristics.Antibodies can be produced in response to antigenic stimuli including,but not limited to, exposure to foreign proteins, microorganisms, andtoxins. When the panel is contacted with a sample containing at leastone antibody specific to an antigen in the panel, an immunocomplex formsbetween the antigen and the antibody specific for the antigen. One ofordinary skill in the art can assess antigen-antibody immunocomplexformation by techniques commonly used in the art. Examples of suitabletechniques to assess immunocomplex formation include phage immunoblot,ELISA, and radioimmunoassay. See, e.g., (Dubovsky et al., J. Immunother.30:675-683 (2007), incorporated herein by reference as if set forth inits entirety). Samples suitable for use with the method include tissues,cells, and bodily fluids. Preferably, the sample is serum.

In a preferred embodiment, at least about 38% of individuals who haveprostate cancer and at least about 33% of individuals who haveprostatitis are immunoreactive to at least one antigen of the panel. Inanother preferred embodiment, at least about 10% of individuals who haveprostate cancer and at least about 9% of individuals who haveprostatitis are immunoreactive to at least three antigens of the panel.In still another preferred embodiment, antigens of the panel areselected based on their capacity in aggregate to elicit an immuneresponse in at least about 33% of individuals with prostatitis orprostate cancer.

Prostate cancer-relevant cancer-testis antigens (CTA) normally expressedonly in MEW class I-deficient germ cells but aberrantly expressed insolid tumors of various histologic types, are suitable for use in thepanel. Suitable CTA antigens include, but are not limited to, CTA1,CTA2, CTA3, CTA4, CTA5, CTA6, CTA7, CTA8, CTA9, CTA10, CTA11, CTA12,CTA13, CTA14, CTA15, CTA16, CTA17, CTA18, CTA19, CTA20, CTA21, CTA22,CTA23, CTA24, CTA25, CTA26, CTA27, CTA28, and CTA29. Antigens identifiedin prostate cancer individuals treated with androgen deprivation therapy(ADT) are also suitable for use in this panel. Suitable ADT antigensinclude, but are not limited to, ADT1, ADT2, ADT3, ADT4, ADT5, ADT6,ADT7, ADT8, ADT9, ADT10, ADT11, ADT12, ADT13, ADT14, ADT15, ADT16,ADT17, ADT18, ADT19, ADT20, ADT21, ADT22, ADT23, ADT24, ADT25, ADT26,ADT27, and ADT28. Antigens identified in prostate cancer individualstreated with non-ADT prostate cancer therapies (PCA) are also suitablefor use in this panel. Suitable PCA antigens include but are not limitedto, PCA1, PCA2, PCA3, PCA4, PCA5, PCA6, PCA7, PCA8, PCA9, PCA10, PCA11,PCA12, PCA13, PCA14, PCA15, PCA16, PCA17, PCA18, PCA19, PCA20, PCA21,PCA22, PCA23, PCA24, PCA25, PCA26, PCA27, PCA28, PCA29, and PCA30.Alternatively, antigens identified in subjects with chronic prostatitis(PRO) are suitable for use in this panel. Suitable PRO antigens include,but are not limited to, PRO1, PRO2, PRO3, PRO4, PRO5, PRO6, PRO7, PRO8,PRO9, PRO10, PRO11, PRO12, PRO13, PRO14, PRO15, PRO16, PRO17, PRO18,PRO19, PRO20, PRO21, PRO22, PRO23, PRO24, PRO25, PRO26, PRO27, PRO28,PRO29, PRO30, PRO31, PRO32, PRO33, PRO34, PRO35, PRO36, PRO37, PRO38,PRO39, PRO40, and PRO41. Exemplary conditions for preparing antigenshaving epitopes specifically recognized by antibodies specific to theepitopes, are well-known by those of ordinary skill in the art.

In a preferred embodiment, the panel includes ADT14, CTA3, CTA23, PCA6,PCA8, PCA10, PCA16, PRO5, PRO10, PRO12, PRO17, PRO18, PRO19, PRO20,PRO24, PRO25, PRO35, CTA27, PCA21, PRO40, PCA2, PRO34, and PRO32.Omitting one or more of these antigens from the panel might affect thepanel's sensitivity. Similarly, adding one or more antigen to this panelmight affect the panel's sensitivity. However, a skilled artisanunderstands that increasing or decreasing the number of antigens in thispreferred panel might still provide an acceptable panel of antigens foridentifying candidates for further prostate cancer examination.

In another preferred embodiment, the panel includes antigens that have ahigh likelihood of eliciting immunoresponses in subjects who arecandidates for further prostate cancer examination, relative to subjectswho are not candidates for further prostate examination, as measured bypositive likelihood ratio. In some of these preferred embodiments,antigens of the panel have a positive likelihood ratio of at least 4(sensitivity/(1-specificity).

In another embodiment, the present invention provides a method foridentifying individuals as candidates for further prostate cancerexamination. In the method, a panel is contacted with a samplecontaining one or more antibodies under conditions suitable forantigen-antibody immunocomplex formation. The skilled artisan knowsexemplary conditions to allow high-affinity antigen-antibody binding(e.g., Dubrovsky et al., 2007 supra). In the method, presence of animmunocomplex indicates that the individual from whom the sample wasobtained is a candidate for further prostate cancer examination.

The disclosed methods can be performed in a single or multi-well formatin which each one of the plurality of antigens is placed in a separatewell of a multi-well assay plate or, alternatively, in a singlecontainer format in which the entire panel of antigens is placed in asingle well. The method may be performed in a qualitative format todetect the presence or absence of immunocomplexes, or in a quantitativeformat to provide a quantitative measurement of the antibodies in asample. Exemplary conditions used to assay antigens of the inventivepanel are well-known by those of ordinary skill in the art.

In another embodiment, the antigen panel can be provided as part of akit containing a carrier or medium suitable for antigen storage.

The invention will be more fully understood upon consideration of thefollowing non-limiting Examples.

EXAMPLES Example 1: Diagnostic Evaluation of Antibody Responses toProstate Cancer-Associated Antigens

Multiple antigens were evaluated to identify a subset of immunoreactiveantigens in humans with prostate disease. IgG responses were evaluatedin subjects with prostate cancer, chronic prostatitis, and in menwithout prostate disease using phage immunoblot methodology. Resultsindicated that subjects with prostate cancer or prostatitis haveantibodies specific for multiple common antigens: of 128 proteinstested, a subset containing 23 proteins was identified to whichantibodies were detected in 38% of subjects with prostate cancer and 33%subjects with prostatitis versus 6% of controls. These results suggestan association between inflammatory conditions of the prostate andprostate cancer, and suggest that antibody responses to a panel ofcommonly-recognized prostate antigens can be used to identify subjectsat risk for prostate cancer.

Materials and Methods

High Throughput Immunoblot (HTI):

Phage immunoblot analysis was performed essentially as described byDubovsky et al., J Immunother 30:675-683 (2007), incorporated byreference herein as if set forth in its entirety. Briefly, panels of 128lambda phage encoding unique prostate-associated antigens were assembled(Table I). Phage included those antigens initially identified inindividuals with prostate cancer or chronic prostatitis by SEREX (Dunphyet al., 2004 supra; Dunphy et al., 2005, supra; Morse and McNeel, 2010supra; Hoeppner et al., 2006 supra), or were constructed to expressspecific genes of interest based on prior studies (Dubovsky et al., 2007supra; Olson and McNeel, Prostate 67:1729-1739 (2007) incorporatedherein by reference as if set forth in its entirety). The followingantigens were included in the panel: 29 cancer-testis antigens (CTA), 41antigens identified in individuals with chronic prostatitis (PRO), 28antigens identified in individuals treated with androgen deprivation(ADT), and 30 antigens identified in individuals treated with othertherapies (PCA).

100,000 pfu lambda phage each carrying a gene that encodes one of the128 antigens of the panel were robotically spotted in triplicate in a16×24 array onto lawns of E. coli (XL-1 blue strain) growing inagar-containing OmniTray plates. Replicates for individual antigens werestaggered in position across the array to account for regional variationon individual filters. For initial studies, phage encoding humanimmunoglobulin G (IgG) were included as a positive control and emptyphage constructs were similarly included as a negative control. Plateswere allowed to dry at room temperature for 20 minutes and incubated at37° C. for 4 hours after which nitrocellulose membranes suffused with10-mM isopropyl ß-D-thiogalactopyranoside (IPTG) (Fisher Scientific,Pittsburgh, Pa.) were overlaid. Plates were incubated at 37° C.overnight to allow for recombinant expression of the antigen encoded bythe protein. After 16-20 hours, membranes were removed, washed twice inTBST (50 mM Tris pH 7.2, 100 mM NaCl, 0.5% Tween-20) for 10 minutes andonce in TBS (50 mM Tris pH 7.2, 100 mM NaCl) for an additional 10minutes. Membranes were blocked in blocking solution (TBST+1% BSA), andincubated at 4° C. with human sera (diluted 1:100 in blocking solution)overnight. Membranes were washed the following day and blocked prior toincubation with a mouse anti-human IgG antibody conjugated to alkalinephosphatase (Sigma, St. Louis, Mo.) to detect human IgG. Membranes werewashed again and incubated in 0.3 mg/mL nitro blue tetrazolium chloride(NBT) (Fisher Scientific) and 0.15 mg/mL 5-bromo 4-chloro3-indoylphosphate (BCIP) (Fisher Scientific) in 100-mM Tris 9.5, 100-mMNaCl, and 5-mM MgCl₂ to detect immunoreactivity. Membranes were washedwith large volumes of deionized water and dried at room temperatureprior to evaluation.

Membranes were scanned using a color image scanner and the digitalformat aligned with a 16×24 grid using densitometry software (ImageQuantTL, Amersham Biosciences, GE Healthcare Life Sciences, Piscataway,N.J.). For initial studies, immunoreactivity was quantified by measuringthe density at each spot; values of replicates for individual antigenswere averaged. Background correction was then made by subtracting theaverage of empty phage construct replicate densities on individualmembrane and normalized by dividing by the average of IgG positivecontrol replicate densities on each membrane. Transformation ofdensitometry data resulted in density values for individual antigensrelative to a negative control and a positive control (set at 0.0 and1.0 respectively). For subsequent studies, immunoreactivity was judgedas “positive” or “negative” by visual inspection, as previouslydescribed (Dunphy et al., J. Clin. Immunol. 24:492-501 (2004),incorporated herein by reference as if set forth in its entirety;Dubovsky et al., 2007 supra). Briefly, four independent individualslooked at each membrane and scored individual spots as immunoreactive ornot reactive by visually comparing each spot to a negative control.Antigens for which 0-1 of replicates determined immunoreactive withindividual sera were defined as negative for immunoreactivity, and forwhich 2-3 of the replicates determined immunoreactive were defined aspositive.

TABLE I One embodiment of the antigen panel. Des- SEQ igna- GenBank IDtion Accession NO: Gene name PRO1 NM_006423.1 1 Rab acceptor PRO2NM_007124.1 2 U-trophon PRO3 NM_000484.1 3 Amyloid Beta (4) precursorprotein PRO4 NM_002709.1 4 Protein Phosphatase 1 PRO5 AC005822.1 5hRPK.209-J-20 DNA from chromosome 17 PRO6 AF308301.1/ 6NY-BR-87/Ribosomal protein S26 NM_030811.2 PRO7 NM_014761.1 7 KIAA0174from chromosome 16 PRO8 AF273042.1 8 Cutaneous T cell lymphoma tumorantigen sel-1 PRO9 AK096728.1 9 FLJ39409 Cdna PRO10 NM_001747.1 10Macrophage capping protein gelsolin-like (CAPG) PRO11 NM_000717.2 11Carbonic anhydrase IV PRO12 AL136040.5 12 BAC C-2506P8 from chromosome14 PRO13 AC084864.4 13 RP11-738B7 DNA from chromo- some 7 PRO14NM_001813.1 14 Centromere protein E PRO15 M27274.1 15 Prostate specificantigen (PSA) PRO16 NM_012116.2 16 Cas-Br-M ecotropic retroviraltransforming sequence c (CBLC) PRO17 BC006286.1 17 Dual specificityphosphatase 12 PRO18 NM_144767.1/ 18 Protein kinase A anchor protein 13/BC017368.1/ lymphoid blast crisis oncogene/ AF126008.1 breast cancernuclear receptor- binding auxiliary protein (BRX) PRO19 AC073879.7 19BAC RP11-752K22 from chromo- some 2 PRO20 AB017363.1 20 Frizzled-1 PRO21AL365273.25 21 RP11-429G19 DNA from chromo- some 10 PRO22 XM_047011.2 22o-fucosyltansferase PRO23 NM_017582.3 23 NICE5 PRO24 NM_014190.1 24Adducin 1 PRO25 AL138752.5 25 RP11-3J10 on chromosome 9 PRO26 BC024007.126 Chitobiase PRO27 AK001572.1 27 FLJ10710 cDNA PRO28 AL356915.19 28RP11-3J10 on chromosome 13 PRO29 NM_006117.1/ 29 Peroxisomal D3, D2enoyl CoA AF257175.1 isomerase/Hepatocellular carci- noma-associatedantigen 64 PRO30 XM_033511.8 30 Helicase with SNF2 domain PRO31AF039689.1/ 31 NY-CO-7/STUB1/CLL-associated XM_083939.1/ antigen KW-8AF432221.1 PRO32 AC021558.10 32 RP11-746L20 DNA from chromo- some 8PRO33 NM_031946.2 33 Centaurin gamma 3 PRO34 BC034250.1 34 Pituitarytumor-transforming 1 interacting protein PRO35 AC069506.14 35 BACRP11-321G3 PRO36 AC011489.6 36 CTB-179K24 DNA on chromo- some 19 PRO37NM_032415.2 37 Caspase domain recruitment (CARD11) PRO38 NM_003379.3 38Cytovillin 2 PRO39 BC029529.1 39 Beta tubulin PRO40 L07872.1 40Recombination signal binding protein (RBPJK) PRO41 NM_006455.1 41Nucleolar autoantigen/MAD-Pro- 34 CTA1 NM_004988 42 Mage A1 CTA2BC007343 43 SSX-2 CTA3 AJ003149 44 Ny-ESO1 CTA4 NM_021123 45 Gage 7 CTA5U90841 46 SSX-4 CTA6 BC015020 47 NXF-2 CTA7 BC022011 48 TPX 1 CTA8BC009538 49 Xage 1 CTA9 BC002833 50 Lage 1 CTA10 BC010897 51 Page 1CTA11 BC081566 52 Mage E1 CTA12 BC054023 53 Span XC CTA13 BC064547 54Adam 2 CTA14 BC037775 55 TSP 50 CTA15 BC034320 56 NY-SAR 35 CTA16BC022064 57 Fate 1 CTA17 BC009230 58 Page 5 CTA18 BC023635 59 Lip1 CTA19BC032457 60 SPA17 CTA20 BE387798 61 Mage A8 CTA21 BE897525 62 Mage B1CTA22 BC026071 63 Mage B2 CTA23 BC017723 64 Mage A4 CTA24 BC001003 65SSX-1 CTA25 BC069397 66 Gage 2 CTA26 BC069470 67 Gage 4 CTA27 BC01680368 Mage A3 CTA28 NM_002762 69 MAD-CT-1 CTA29 AK097414 70 MAD-CT-2 PCA1NM_025161.4 71 Chromosome 17 gene contig. PCA2 NM_000972.2 72 Ribosomalprotein L7a PCA3 NM_152636.2 73 Chromosome 12 gene contig. PCA4NM_001134194.1 74 Prostatic acid phosphatase PCA5 NM_003291.2 75Tripeptidyl peptidase II PCA6 NM_006690.3 76 Matrix metallopeptidase 24PCA7 NM_000990.4 77 Ribosomal protein L27a PCA8 NM_002652.2 78Prolactin-induced protein PCA9 NM_005349.2 79 Immunoglobulin Kappa Jregion PCA10 NM_005817.2 80 Mannose-6-phosphate receptor binding protein1 PCA11 NM_013267.2 81 Glutaminase 2 PCA12 NM_018979.2 82 WNK lysinedeficient protein kinase 1 PCA13 NM_020718.3 83 Ubiquitin specificpeptidase 31 PCA14 NM_003007.2 84 Semenogelin I, transcript variant 1PCA15 NM_022735.3 85 Acyl-coenzyme A binding domain containing 3 PCA16NM_001040284.1 86 PAP associated domain PCA17 NM_020187.2 87 Chromosome3 gene contig. PCA18 NM_002712.1 88 Protein phosphatase 3, regulatorysubunit 7 PCA19 NM_014220.2 89 Transmembrane 4 L6 family mem- ber 1PCA20 NM_001135592.1 90 Ribosomal protein S27a PCA21 NM_012401.2 91Plexin B2 PCA22 NM_000985.3 92 Ribosomal protein L17 PCA23 NM_080608.393 Chromosome 20 gene contig. PCA24 NM_005165.2 94 Aldolase C PCA25NM_000969.3 95 Ribosomal protein L5 PCA26 NM_001958.2 96 Eukaryotictranslation elongation factor 1 alpha 1 PCA27 NM_025108.2 97 Chromosome16 gene contig. PCA28 NM_015358.2 98 Zinc-finger protein, CW type withcoiled-coil domain 3 PCA29 NM_001130410.1 99 Acetyl-coenzyme Aacyltransfer- ase 1 PCA30 NM_000044.2 100 Androgen receptorligand-binding domain ADT1 NM_004759.3 101 Mitogen-activated proteinkinase- activated protein kinase 2 ADT2 NM_001103.2 102 Actinin alpha 2ADT3 NM_002518.3 103 Neuronal PAS domain protein 2 (NPAS2) ADT4NM_033138.2 104 Caldesmon 1 (CALD1) ADT5 NM_001459.2 105 fms-relatedtyrosine kinase 3 ligand ADT6 NM_022474.2 106 Membrane protein,palmitoylated 5 (MAGUK p55 subfamily member 5) ADT7 NM_006022.2 107TSC22 domain family, member 1 (TSC22D1) ADT8 NM_001001522.1 108Transgelin ADT9 NM_022758.4 109 Chromosome 6 gene contig ADT10NM_002695.3 110 polymerase (RNA) II (DNA directed) polypeptide E(POLR2E) ADT11 NM_001018160.1 111 NEDD8 activating enzyme subunit 1(NAE1) ADT12 NM_015313.1 112 Rho guanine nucleotide exchange factor 12(ARHGEF12) ADT13 NM_152374.1 113 Chromosome 1 gene contig ADT14NM_001100873.1 114 Chromosome 16 gene contig ADT15 NM_001136204.1 115Regulator of chromosome conden- sation 2 (RCC2) ADT16 NT_006713.15 116Chromosome 5 gene contig (splicing factor, arginine/serine- rich 12)ADT17 NM_025157.3 117 Paxillin (PXN) ADT18 NM_016143.2 118 NSFL1 (p97)cofactor (p47) NSFL1C ADT19 NM_004913.2 119 Chromosome 16 gene contigADT20 NM_207356.2 120 Chromosome 1 gene contig ADT21 NM_003104.4 121Sorbitol dehydrogenase (SORD) ADT22 NT_006316.16 122 Chromosome 4 genecontig (solute carrier family 2, facilitated glucose transporter) ADT23NM_013336.3 123 Sec 61 alpha 1 subunit ADT24 NM_002474.2 124 Myosinheavy chain 11 (MYH11) ADT25 NR_002819.2 125 Metastasis associated lungadenocarcinoma transcript 1 (MALAT1) ADT26 NM_001012321.1 126 Similar tolamanin receptor 1 (ribosomal protein SA (RPSA), from chromosome 19)ADT27 NT_009952.14 127 Chromosome 13 gene contig (heparan-sulfate6-O-sulfotrans- ferase 3) ADT28 NM_021239.2 128 RNA binding motifprotein 25 (RBM 25)

Statistical Analyses:

Data collected from preliminary studies analyzing individuals withcastrate-resistant prostate cancer and normal male blood donors wereplotted as relative density values representing detection of antibodiesspecific to 125 prostate cancer-associated antigens for individualindividuals. Median relative density values for individual antigens werecalculated in both castrate-resistant prostate cancer samples and normalcontrol samples and the non-parametric Wilcoxon Rank Sum test was usedto compare the medians of the two groups. The Benjamini-HochbergFalse-Discovery Rate (FDR) method for multiple testing was used tocontrol the type I error (Benjamini et al., J. Roy. Stat. Soc. B(Method) 57:289-300 (1995) incorporated by reference herein as if setforth in its entirety). The frequencies of IgG responses were comparedbetween populations using Fisher's exact test. Receiver Operating Curve(ROC) analysis was performed to identify a subset of prostatecancer-associated antigens with the highest predictive value fordetecting prostate cancer cases when compared to normal controls. Thepositive likelihood ratio value was used to quantify the predictivevalue for individual markers. The proportions of individuals with atleast one positive IgG response and the proportions of individuals withat least three positive IgG responses in the subset of predictivemarkers were compared between populations by performing logisticregression analysis where population groups were included as factors.Dunnett's multiple testing procedure was used to compare the proportionsbetween the individual populations and the control (normal) group. Allp-values are two-sided, with p<0.05 indicating statistically significantdifferences. The data analysis was performed using SAS® version 9.2software (SAS Corp., Cary, N.C.).

Individual Populations:

Sera were obtained from individuals with chronic prostatitis (n=45,median age 42, age range 19-62), prostate cancer (n=151, median age 67,age range 44-86), and men without a history of known prostate disease(n=78, median age 32, age range 18-62). Among the individuals withprostate cancer, 18 were collected from individuals at the time ofdiagnosis prior to definitive radiation treatment or prostatectomy(pretreatment), 32 were collected from individuals after definitivetreatment without evidence of disease recurrence (limited stage), 44were collected from individuals with metastatic disease recurrence onandrogen deprivation therapy (androgen-dependent), and 57 were collectedfrom individuals with castrate-resistant, metastatic disease. Sera werealso obtained from male individuals with other cancers (melanoma, n=4;renal, n=17; testicular, n=5; other, n=5; median age 58, age range30-74) for use as controls. All subjects gave written InstitutionalReview Board-approved consent for the use of their blood forimmunological studies. Blood was collected at the University ofWisconsin-Madison Hospital and Clinics (Madison, Wis.) or at theUniversity of Washington Medical Center (Seattle, Wash.), and sera werestored in aliquots at −20° C. to −80° C. until used for analysis.

Results

Individuals with Prostate Cancer have Higher Frequencies of DetectableAntibodies Specific for Prostate Cancer-Associated Antigens than Menwithout Cancer.

To determine if specific antigens are commonly recognized in the sera ofindividuals with prostate cancer, and if a particular set of antigens ismore frequently recognized than other antigen sets, phage encoding 125unique, prostate cancer-associated antigens obtained from prior studies(Table I) were spotted in replicate onto bacterial lawns, transferred tonitrocellulose membranes, and probed with individual sera samples, asillustrated in FIG. 1. IgG specific for phage-encoded plaques weredetected using a mouse anti-human IgG antibody, immunoreactivity wasquantified by densitometry, and then normalized to internal controls(0=reactivity equivalent to phage not encoding a protein; 1=reactivityequivalent to phage encoding human IgG). Immunoreactivity to multipleantigens was detected in the individual population sampled (i.e.,individuals with castrate-resistant metastatic prostate cancer) that wasabsent in the sera of 25 healthy male blood donors (FIG. 2A-H). Of the125 antigens evaluated, median antibody responses to 27 (22%) antigenswere significantly higher in the sera of individuals with prostatecancer compared with controls (p<0.05, Wilcoxon Rank Sum test, FIG.2A-H). Of these 27 antigens, 22 were previously identified inindividuals with chronic prostatitis.

Antibody Responses to Antigens of the Antigen Panel are Evident inIndividuals with Early and Late Stages of Prostate Cancer and inIndividuals with Prostatitis.

To determine if recognition of particular antigens distinguishesindividuals with prostate cancer from individuals with prostatitis orfrom men without known prostate disease, sera from a larger populationof healthy male control blood donors (n=53), individuals with chronicprostatitis (n=45), and individuals with different stages of prostatecancer (n=126) were analyzed. Serum samples were obtained from men withnewly diagnosed prostate cancer (pre-treatment, n=18), individuals withtreated disease and no evidence of recurrence (n=32), individuals withmetastatic disease responsive to androgen deprivation therapy (n=44),and additional individuals with castrate-resistant metastatic disease(n=32). In addition, the panel of antigens was modified to exclude twoantigens (Pro39 and ADT28) not immunologically recognized in the initialscreen, and to include phage containing genes that encoded threeadditional antigens (Mad-Pro-30, SEQ ID NO: 30; Mad-Pro-34, SEQ ID NO:34; and the androgen receptor ligand-binding domain, SEQ ID NO:129) thathad previously been identified as immunologically recognized antigens(Dunphy et al., 2004 supra; Olson and McNeel, 2007 supra). To accountfor background reactivity of individual membranes and variability inplaque immunoreactivity, each of the replicate individual plaques wasvisually scored as immunoreactive or not.

As demonstrated in FIG. 3A, immunoreactivity to multiple antigens wasidentified. FIG. 3A shows a heatmap depicting relative immunoreactivityfor each antigen, grouped by the study from which the antigens wereoriginally identified, and for each individual or control group. Spotsare graded in color intensity from 3 of 3 replicates visuallyimmunoreactive (white), to 2 of 3 (grey), to not immunoreactive (black).Overall, however, responses to at least one antigen were not morefrequently observed in the individual population ( 72/126, 57%) orprostatitis population ( 28/45, 62%) compared with the controlpopulation ( 22/53, 42%; p=0.11 and p=0.08, respectively). We identifiedno single antigen to which immunoreactivity was significantly morefrequent in the individual population than in the control population.Instead, 23 antigens with high (>4) positive likelihood ratio valueswere identified based on a ROC analysis of IgG responses to antigensamong prostate cancer individual samples compared to control individualsamples (Table II). FIG. 3B-F shows a subset analysis for the 23antigens with the highest positive likelihood ratio values whencomparing prostate cancer cases to normal control cases.

TABLE II One embodiment of the antigen panel. Shown is the subset oflambda phage-encoded antigens with the highest specificity for prostatecancer based on receiver operating curve analysis (see also FIG. 3B-F).Antigens with a positive likelihood ratio > 4(sensitivity/(1-specificity)) are included, and the positive likelihoodratio is shown. Positive Des- Likeli- SEQ igna- hood ID tion Ratio NO:Gene name ADT14 ∞ 114 Chromosome 16 gene contig CTA3 ∞ 44 NY-ESO-1 CTA23∞ 64 Mage A4 PCA6 ∞ 76 Matrix metallopeptidase 24 PCA 8 ∞ 78Prolactin-induced protein PCA 10 ∞ 80 Mannose-6-phosphate receptorbinding protein 1 PCA 16 ∞ 86 PAP-associated domain PRO5 ∞ 5hRPK.209-J-20 DNA from chromosome 17 PRO 10 ∞ 10 Macrophage cappingprotein gelsolin-like (CAPG) PRO 12 ∞ 12 BAC C-2506P8 from chromosome 14PRO 17 ∞ 17 Dual specificity phosphatase 12 PRO 18 ∞ 18 Protein kinase Aanchor protein 13/lymphoid blast crisis oncogene/breast cancer nuclearreceptor-binding auxiliary protein (BRX) PRO 19 ∞ 19 BAC RP11-752K22from chromosome 2 PRO 20 ∞ 20 Frizzled-1 PRO 24 ∞ 24 Adducin 1 PRO 25 ∞25 RP11-3J10 on chromosome 9 PRO 35 ∞ 35 BAC RP11-321G3 CTA27 8.4 68Mage A3 PCA 21 8.4 91 Plexin B2 PRO 40 6.7 40 Recombination signalbinding protein (RBPJK) PCA 2 5.5 72 Ribosomal protein L7a PRO 34 5.4 34Pituitary tumor-transforming 1 interacting protein PRO 32 4.2 32RP11-746L20 DNA from chromosome 8

Antibody Responses to Prostate Tissue-Associated Antigens OccurIrrespective of the Stage of Prostate Cancer.

FIG. 4 shows the proportion and standard error of subjects in eachpopulation recognizing at least one (FIG. 4A) or at least three (FIG.4B) of the antigens identified in Table II. Asterisks denote higherfrequency compared to the control population (p<0.05, computed bylogistic regression analysis and Dunnett's method for multiplecomparisons). A subset analysis showed that the proportion ofindividuals with a detectable antibody response to at least one of the23 antigens was significantly higher in the prostate cancer individualpopulation when compared to the control population (FIG. 4A), that is,48/126 (38%) versus 3/53 (6%) (p<0.001). Antibody responses detected insera from prostatitis individuals ( 15/45, 33%) were significantlyhigher than those detected in the control population (p=0.003). Antibodyresponses detected in sera from prostatitis individuals were notsignificantly different from those detected in the prostate cancerindividual population (FIG. 4A). As shown in FIG. 4B, the presence ofimmunoreactivity to three or more of the 23 antigens was more frequentin individuals with prostate cancer ( 12/126, 10%) and individuals withprostatitis ( 4/45, 9%) relative to male control blood donors ( 1/53,2%).

Antibody Responses to Prostate-Associated Antigens are Uncommon in Serafrom Male Individuals with Non Prostate Malignancies.

The majority of antigens recognized by prostate cancer and prostatitisindividuals are not prostate-specific in terms of expression, suggestingthat the responses detected could be signatures of non-prostate-specificinflammatory conditions. To test this possibility, antibody responses toa panel of 126 antigens were evaluated using sera obtained from maleindividuals (n=31) with other non-prostate cancers (renal cell cancer,n=17; testicular cancer, n=5; melanoma, n=4; head and neck cancer, n=1;GI stromal tumor, n=1; bladder cancer, n=1; non-small cell lung cancer,n=1; carcinoid tumor n=1). FIG. 5 shows a heatmap depictingimmunoreactivity for each antigen, grouped by the study from which theywere originally identified, and for each individual tumor type. Spotswere scored positive (white) if visually immunoreactive in at least 2 of3 replicates. As shown in FIG. 5, while antibody responses weredetectable to at least one of the 126 antigens in 7/31 sera samples(23%), only one response in 1/31 sera samples (3%) was detected to oneof the 23 prioritized antigens. This result was not statisticallydifferent in frequency from responses identified in the non-cancercontrol population. The one response identified was to the Pro32antigen, which exhibited the lowest positive likelihood ratio (TableII).

DISCUSSION

Antigenic Targets of a Prostate-Associated Immune Response areRecognized in Populations of Individuals with Prostatitis and ProstateCancer.

Immune responses to antigens originally identified in individuals withchronic prostatitis are also recognized in individuals with advancedprostate cancer relative to controls, suggesting that many “prostatitisantigens” represent true immunologically recognized antigens of theprostate, many of which coincide with antibody recognition inindividuals with prostate cancer. Specific antibody responses to atleast one member of a subset of 23 of these antigens were detectable insera of individuals with prostate cancer or clinical prostatitis, butwere rare in sera from male subjects with other types of cancer and inmale subjects without prostate cancer or prostatitis.

These results, while not suggesting causality, suggest an associationbetween chronic inflammatory conditions, such as prostatitis, andprostate cancer, or at least suggest that targets of aprostate-associated immune response can be recognized in thesedisorders. These results further suggest that the prostate expresses aset of antigens recognized immunologically by multiple individuals. Theevaluation of antibody responses to prostate-associated antigens canpotentially identify individuals in a premalignant inflammatory state atrisk for developing prostate cancer. To further test this potential,sera obtained from individuals without a history of prostatitis but whodeveloped prostate cancer within several years can be screened using theset of antigens identified herein.

Advantages of the phage immunoblot approach described here are the easeof transfection and protein expression in bacteria, particularly fornovel proteins for which there are no available reagents, and theability to simultaneously evaluate antibody responses to multipleantigens simultaneously from a single sample.

It is understood that certain adaptations of the invention described inthis disclosure are a matter of routine optimization for those skilledin the art, and can be implemented without departing from the spirit ofthe invention, or the scope of the appended claims. All publications andpatents mentioned in the above specification are herein incorporated byreference. Various modifications and variations of the described methodand system of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. It isunderstood, however, that examples and embodiments of the presentinvention set forth above are illustrative and not intended to confinethe invention. The invention embraces all modified forms of the examplesand embodiments as come with the scope of the following claims.

We claim:
 1. A method of identifying a human with prostate cancer, prostatitis or at risk of developing prostate cancer comprising the steps of: (a) contacting a serum sample obtained from the human with a panel comprising antigens encoded by SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:40, SEQ ID NO:44, SEQ ID NO:64, SEQ ID NO:68, SEQ ID NO:72, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:86, and SEQ ID NO:91, the encoded antigens being immobilized on one of a membrane, a plate, and a filter, and having a positive likelihood ratio of at least 4, wherein the sample comprises at least one antibody; and (b) identifying the human with prostate cancer, prostatitis or at risk of developing prostate cancer if at least one immunocomplex forms between the at least one antibody and at least one antigen of the panel.
 2. The method of claim 1, wherein the panel further comprises an antigen encoded by a nucleic acid having a sequence selected from the group consisting of SEQ ID Nos: 1-4, 6-9, 11, 13-16, 21-23, 26-31, 33, 36-39, 31-43, 45-63, 65-67, 69-71, 73-75, 77, 79, 81-85, 87-90, and 92-128.
 3. The method of claim 1, the panel consisting of antigens encoded by SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:40, SEQ ID NO:44, SEQ ID NO:64, SEQ ID NO:68, SEQ ID NO:72, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:86, and SEQ ID NO:91.
 4. The method of claim 1, wherein each antigen is expressed by a phage and attached to the membrane, plate or filter via the phage.
 5. The method of claim 1, wherein at least three immunocomplexes between antibodies in the sample and antigens of the panel are detected. 